Project Summary Drug resistance in malaria is one of the most formidable barriers to treatment, control and elimination. Putative polymorphisms in drug resistance transporters have been identified in Plasmodium vivax, the most widespread of all of the species causing human malaria. However, the ability of these polymorphisms to confer chloroquine resistance, or the magnitude of the resistance conferred by these mutations remains unknown. This gap in our knowledge is largely due to the absence of an in vitro culture system, robust drug assays, and reverse genetics for the study of P. vivax. In this proposal, we seek to understand the impact of polymorphisms in putative drug resistance transporters that have been identified in molecular epidemiological studies of P. vivax. We will take advantage of the macaque parasite Plasmodium knowlesi for the analysis of naturally occurring P. vivax polymorphisms, due to its close phylogenetic relatedness to P. vivax, and its versatility as a system for reverse genetics and drug susceptibility assays. We will utilize CRISPR/Cas9-based approaches directly in P. knowlesi for the functional analysis of the P. vivax drug transporter genes and their polymorphisms. We recently culture-adapted a P. knowlesi parasite line to grow in human red blood cells, which is ideally suited for our studies. Reverse genetic analyses of specific drug transporter polymorphisms of P. vivax will be critical to our understanding of their relevance to antimalarial resistance, the development of strategies for limiting the spread or reversal of drug resistance, and their validation as drug resistance markers in population level studies and surveillance for control and elimination measures. Our work will also establish P. knowlesi as a powerful heterologous platform for the study of critical genes involved in many processes relevant to P. vivax.